Dibromoacetonitrile (DBAN) is a disinfection by-product classified as a potential human and animal carcinogen. This study aimed at investigating the ability of myeloperoxidase (MPO) to oxidize DBAN to cyanide (CN^–) in vitro. Detection of CN^– served as a marker for the possible generation of free radical intermediates implicated in DBAN-induced toxicity. Optimum conditions for the oxidation of DBAN to CN^– were characterized with respect to pH, temperature, and time of incubation as well as DBAN, MPO, potassium chloride, and hydrogen peroxide (H₂O₂) concentrations in incubation mixtures. Maximum reaction velocity and Michaelis–Menten constant were assessed. Addition of sodium hypochlorite to the reaction mixtures significantly enhanced the rate of the reaction. Addition of the MPO inhibitors, sodium azide, 4-amino benzoic acid hydrazine, or indomethacin to the reaction mixtures significantly decreased the rate of DBAN oxidation. Inclusion of the antioxidant enzyme superoxide dismutase in the incubation mixtures significantly decreased the rate of reaction. Inclusion of the sulfhydryl compounds as reduced glutathione, N-acetylcysteine, d-penicillamine, or l-cysteine enhanced the rate of DBAN oxidation. These results demonstrate the ability of MPO/H₂O₂/chloride ion system to oxidize DBAN to CN^– and provide insight for the elucidation of DBAN chronic toxicity.